Toward an Integrated Structural Model of the 26S Proteasome

The 26S proteasome is the end point of the ubiquitin-proteasome pathway and degrades ubiquitylated substrates. It is composed of the 20S core particle (CP), where degradation occurs, and the 19S regulatory particle (RP), which ensures substrate specificity of degradation. Whereas the CP is resolved to atomic resolution, the architecture of the RP is largely unknown. We provide a comprehensive analysis of the current structural knowledge on the RP, including structures of the RP subunits, physical protein-protein interactions, and cryoelectron microscopy data. These data allowed us to compute an atomic model for the CP-AAA-ATPase subcomplex. In addition to this atomic model, further subunits can be mapped approximately, which lets us hypothesize on the substrate path during its degradation.The ubiquitin-proteasome pathway is the major route used by eukaryotic cells for the disposal of misfolded or damaged proteins and for controlling the lifespan of proteins (1–3). As a consequence, the ubiquitin-proteasome pathway regulates a plethora of fundamental cellular processes, such as protein quality control, DNA repair, and signal transduction (4). The 26S proteasome is a molecular machine of ∼2.5 MDa that targets polyubiquitylated proteins. It comprises two subcomplexes, the 20S core particle (CP)¹ and one or two copies of asymmetric 19S regulatory particles (RPs), which bind to the end(s) of the barrel-shaped CP.The active sites of the proteasome are located in the CP cavity where proteolytic cleavage of substrates takes place. Electron microscopy (EM) and x-ray crystallography have revealed that the CP is a cylinder consisting of four concentrically stacked rings (5–7): two identical “α”-rings, each assembled of seven homologous proteins, form the outer rings, and two identical “β”-rings, also assembled of seven homologs, form the two inner rings. Proteolysis is confined to the cavity formed by the β-rings, a nanocompartment sequestered from the cytosol.The RPs regulate substrate degradation by (i) binding polyubiquitylated substrates, (ii) subsequently deubiquitylating them, (iii) substrate unfolding, and (iv) opening the “gate” to the CP (8). The RPs consists of six AAA-ATPase subunits and at least 13 non-ATPase subunits. In contrast to the CP, the architecture of the RP subunits remains largely unknown. The problems that hamper structural characterization of the RP are manifold. It has proven difficult to obtain homogeneous, concentrated preparations of 26S proteasomes or RPs because the RP tends to disassociate into heterogeneous subcomplexes during purification and concentration. Moreover, many of the RP subunits likely exhibit a significant degree of structural variability. As a consequence, x-ray crystallographic analysis of the entire RP has not been accomplished to date, and only a few subunit fragments have been amenable to high resolution structure determination.For cryo-EM and protein-protein interaction experiments, the requirements for sample homogeneity are less stringent. Recently, the Drosophila melanogaster 26S proteasome was resolved to ∼20 Å (9). Various proteomics approaches have led to proposals for topological maps of the RP (10–13). The resolution of protein-protein interaction data typically corresponds to the diameters of the proteins or domains found to interact, which are typically far beyond 20 Å. Because of the limited resolution, neither cryo-EM maps nor protein-protein interaction networks are by themselves sufficient to determine the RP architecture (i.e. the localization of the RP subunits in the complex).The integration of atomic models, cryo-EM maps, and protein-protein interaction data is currently the most promising approach to resolve the architecture of the 26S proteasome (14–18). Here, we provide a comprehensive analysis of the current structural knowledge on the RP, including structures of RP subunits, physical protein-protein interactions, and cryo-EM data. Based on these data, we provide a model for the CP-AAA-ATPase subcomplex. Finally, we outline a path toward resolving the architecture of the 26S proteasome by an integrative structure determination approach, which in turn will provide a basis for a mechanistic understanding.